In this K18 Career Enhancement Award in Stem Cell Reseach application, the Candidate (Dr. James Bassuk) proposes to take time from his regular professional responsibilities in order to broaden his scientific background by acquiring new research capabilities. The specifiic new research capability to be learned is the use of embryonic stem cells (ESCs) in regenerative medicine applications such as tissue engineered repair of the urinary bladder and its associated tract. There is a significant need to develop (i) replacement bladders for patients suffering from cancer, interstitial cystitis or neurogenic bladder (spina bifida or multiple sclerosis) and (ii) epithelial tubes for reconstruction of the urethra due to trauma, stricture disease or hypospadias (a common birth defect in boys). The project represents a true career enhancement for him because it brings to his research program, for the first time, an experimental design that focuses on how developmental steps of bladder formation can be studied with a stem cell-based approach. The strategy involves finding the minimum number of ESCs needed to form a teratoma with the goal of redirecting these ESCs to form only one organ type. Embryonic mesenchyme is well established as an inductive tissue which dictates organ-specific programming of epithelial tissues. In Aim 1, the activity of rat embryonic bladder mesenchyme in driving mouse ESCs into functional bladder tissue will be measured by a stereological quantification of urothelial cell terminal differentiation. Under the guidance of Drs. Carol Ware (Mentor), C. Anthony Blau (Co-Mentor), Simon Hayward (Contributor) and Andrew Farr (Contributor), the Candidate will be trained in the proper handling of embryonic stem cells and in setting up xenograft assays where recombinant grafts are implanted under the renal capsule of athymic nude mice. In Aim 2a, human ESCs will be substituted for mouse ESCs. In Aim 2b, mesenchyme from human infant bladder will be substituted for rat embryonic mesenchyme. The source of infant mesenchyme will be the urology service at Seattle Children's Hospital, as procured by Drs. Richard Grady and Thomas Lendvay (Contributors). Thus, the question asked is will human ESCs respond appropriately to bladder mesenchymal developmental control? Over a 2-year project period, the Candidate will devote 75% of his time to train at the Institute for Stem Cell and Regenerative Medicine at the University of Washington. Once the project has been completed, then the Candidate's newly developed expertise will be applied to (i) the further study of mesenchymal factors necessary for developmental control of bladder tissue formation from inducible pluripotent stem cells, (ii) generating new in vitro models of urothelial cell differentiation and (iii) tissue engineered solutions for urologic use. PUBLIC HEALTH RELEVANCE: This project will provide a training experience in stem cell technology for Dr. James Bassuk, an Associate Professor of Urology at the University of Washington. Once the project is over, Dr. Bassuk will apply his newly acquired expertise to developing new solutions to assist the surgeon in repairing the urinary bladder and its associated tract. These efforts are expected to lead to improved outcomes for patients suffering with a non-functional bladder due to cancer, interstitial cystitis, multiple sclerosis or spina bifida.